1. CRISPRa induction of Bdnf mRNA increases spike and burst frequency in hippocampal neurons cultured on microelectrode arrays (MEAs).
CRISPRa induction of Bdnf mRNA increases spike and burst frequency in hippocampal neurons cultured on microelectrode arrays (MEAs). A, CRISPRa induction of Bdnf I and IV increases Bdnf protein quantified by immunoblotting (n = 6 per group; Mann–Whitney U test, U = 0, p = ). B, Primary hippocampal neurons grown on MEAs and transduced with dCas9-VPR and LacZ (top) or Bdnf I and IV (bottom) sgRNAs. mCherry signal indicates successful transduction of sgRNAs in live cultures (right). Scale bar = 100 μm. C, Experimental timeline for viral transduction and MEA recordings. Representative traces (D) and raster plots (E) from 10 units after LacZ (top) or Bdnf I and IV (bottom) targeting. F, The number of active units per well does not change between LacZ and Bdnf I and IV targeted conditions (n = 10–12, unpaired Student’s t test; p = ). G, Action potential frequency across DIV7–DIV11 showing an increase of mean frequency after Bdnf I and IV sgRNA treatment by DIV11, as compared to LacZ sgRNA (n = 57–98 neurons, two-way ANOVA with main effect of sgRNA, F(1,493) = 8.561, p = , Sidak’s post hoc test for multiple comparison). H, Spike frequency at DIV11 for all units ranked from highest to lowest mean frequency showing an increase in activity for the top 1/3 most active units in Bdnf I and IV versus LacZ targeted conditions. I, Burst frequency at DIV11 is increased after Bdnf I and IV versus LacZ targeting (n = 98, unpaired Student’s t test; p = ). All data are expressed as mean ± SEM; *p < 0.05 and **p < The physiological consequences of CRISPRa induction at another gene target (Reln) is shown in Extended Figure 6-1.
Katherine E. Savell et al. eNeuro 2019;6:ENEURO
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